Mammalian cells can shift from a proliferating state to a quiescent state only during a brief window of the cell cycle. Temin, J. Cell. Phys. 78:161 (1971). Thus, depending on their position in the cell cycle, cells deprived of mitogens such as those present in serum will undergo immediate cell cycle arrest, or they will complete mitosis and arrest in the next cell cycle. The transition from mitogen-dependence to mitogen-independence occurs in the mid- to late-G1 phase of the cell cycle. Pardee, Proc. Natl. Acad. Sci. 71:1286 (1974), showed that many different anti-mitogenic signals cause the cell cycle to arrest at a kinetically common point, and further showed that the cell cycle becomes unresponsive to all of these signals at approximately the same time in mid- to late-G1. This point was named the restriction point, or R point.
Time-lapse cinematography of mitotically proliferating single cells has also been used to precisely map the timing of the cell cycle transition to mitogen-independence. This confirmed that mitogen depletion or other growth inhibitory signals cause post-mitotic, early-G1 cells to immediately exit the cell cycle, and that cell cycle commitment (autonomy from mitogenic signals), occurs in mid-G1 (Larsson et al., J. Cell. Phys. 139:477 (1989), and Zetterberg et al., Proc. Natl. Acad. Sci. USA 82:5365 (1985)). Together these observations show that the mitogen-dependent controls on cell proliferation are linked to cell cycle progression.
Transit through G1 and entry into S phase requires the action of cyclin-dependent kinases (Cdks) (Sherr, Cell 79:551 (1994)). Growth inhibitory signals have been shown to prevent activation of these Cdks during G1 (Serrano et al., Nature 366:704 (1993); Hannon and Beach, Nature 371:257 (1994); El-Deiry et al., Cell 75:89 (1993); Xiong et al., Nature 366:701 (1993); Polyak et al., Cell 78:59 (1994); Toyashima and Hunter, ibid., p. 67; Lee et al., Genes & Dev. 9:639 (1995); Matsuoka et al., ibid., p. 650; Koff et al., Science 260:536 (1993)). The catalytic activity of Cdks is known to be regulated by two general mechanisms, protein phosphorylation and association with regulatory subunits (Gould et al., EMBO J. 10:3297 (1991); Solomon et al., ibid., 12:3133 (1993); Solomon et al., Mol. Biol. Cell 3:13 (1992); Jeffrey et al., Nature 376:313 (1995); Morgan, Nature 374:131 (1995)). Among the regulatory subunits, the association of Cdks with inhibitory CKI subunits (Cyclin-dependent Kinase Inhibitors) has been most closely correlated with the effect of mitogen depletion on cell proliferation and Cdk activity.
The CKI directly implicated in mitogen-dependent Cdk regulation is p27Kip1 (Polyak et al., Cell 78:59 (1994); Toyashima and Hunter, ibid., p. 677). The p27 protein accumulates to high levels in quiescent cells, and is rapidly destroyed after quiescent cells are re-stimulated with specific mitogens (Nourse et al., Nature 372:570 (1994); Kato et al., Cell 79:487 (1994)). Moreover, constitutive expression of p27 in cultured cells causes the cell cycle to arrest in G1 (Polyak supra, Toyashima and Hunter, supra).
Gene therapy is proposed for treating and preventing a wide variety of acquired and hereditary diseases, such as infectious diseases, cancer, etc. and relies on the efficient delivery of therapeutic genes to target cells. Most of the somatic cells that have been targeted for gene therapy, e.g., hematopoietic cells, skin fibroblasts and keratinocytes, hepatocytes, endothelial cells, muscle cells and lymphocytes, are normally non-dividing. Retroviral vectors, which are the most widely used vectors for gene therapy, unfortunately require cell division for effective transduction (Miller et al., Mol. Cell. Biol. 10:4239-4242 (1990)). This is also true with other gene therapy vectors such as the adeno-associated vectors (Russell et al., Proc. Natl. Acad. Sci. USA 91:8915-8919 (1994); Alexander et al., J. Virol. 68:8282-8287 (1994); Srivastrava, Blood Cells 20:531-538 (1994)). The majority of stem cells, a preferred target for many gene therapy treatments, are normally not proliferating. Thus, the efficiency of transduction is often relatively low, and the gene product may not be expressed in therapeutically or prophylactically effective amounts. This has led investigators to develop techniques such as pretreatment with 5-fluorouracil, infection in the presence of cytokines, and extending the vector infection period to increase the likelihood that stem cells are dividing during infection, but these have met with limited success.
What is needed in the art is a method for improving the efficiency of gene transfer that is useful for a wide variety of gene therapy applications. For example, what is needed is a means to improve transduction efficiency into a wide variety of vertebrate cells with vectors that can transduce only dividing cells by controlling key molecular events in the cell cycle commitment through the Restriction point and thus cell cycle progression. Quite surprisingly, the present invention fulfills this and other related needs.